Synaptic transmission is responsible for information transfer between neurons and its regulation underlies learning, memory, and many aspects of physiological regulation. N-type and P/Q-type Ca currents through Cav2.1 and Cav2.2 channels, respectively, are responsible for the Ca entry that initiates neurotransmitter release at most conventional fast synapses. Ca entering through presynaptic Ca channels forms a local domain of high Ca concentration that activates exocytosis in the near vicinity. Therefore, synaptic vesicles must dock near presynaptic Ca channels to be efficiently released. Neurotransmitter release is dependent on the third or fourth power of the Ca current through presynaptic Ca channels, so small changes in Ca entry have large effects on synaptic transmission. Synaptic plasticity due to Ca-dependent facilitation and inactivation of synaptic transmission is an important determinant of information coding and transmission. Many neurotransmitters that act through G protein-coupled receptors can inhibit the activity of presynaptic Ca channels and thereby inhibit synaptic transmission. Their inhibition is relieved by strong depolarization or by phosphorylation by protein kinase C. Our results in the present project period have given important new insights into regulation of presynaptic Ca channels by G proteins. We have analyzed the structure-function relationships of SNARE protein binding and its regulation at the synaptic protein interaction site, and we have shown that this site is necessary for reconstitution of synaptic transmission by exogenously expressed Ca channels. In addition, we have discovered a novel mechanism of Ca channel regulation by calmodulin and neuro-specific Ca binding proteins, which are likely to have important roles in short-term synaptic plasticity. In the next project period, we plan to build on these advances to further define the molecular mechanism of Ca channel function and regulation in synaptic transmission. We will determine the sites and mechanisms of action and the diversity of regulation of Cav2.1 channels by neuro-specific calmodulin (CaM)-Iike Ca binding proteins. We will further define the site of interaction of SNARE proteins with the synaptic protein interaction site of Cav2.1 channels and the regulation of SNARE protein binding and Cav2.1 channel function by phosphorylation by protein kinase C (PKC) and Ca/CaM-dependent protein kinase II (CaMKII). Based on this molecular information, we will analyze the functional significance of neuro-specific Ca binding proteins, SNARE proteins, and protein phosphorylation in regulation of synaptic transmission neurons in cell culture. Finally, we will probe the convergent regulation of Cav2.1 channel function and synaptic transmission by Ca-binding proteins, SNARE proteins, and protein phosphorylation. These studies will give important new insight into the mechanism and regulation of synaptic transmission and will further define the function of Cav2.1 channels and their regulation in synaptic plasticity.